Paramagnetic compounds

ABSTRACT

There are provided paramagnetic compounds comprising a paramagnetic metal species chelated by a chelating moiety bound by an amide group to a linker group itself bound by an ester group to a macromolecule, wherein said linker group provides a carbon chain of at least 2 atoms between said amide group and said ester group. The novel compounds are particularly suitable as contrast agents, e.g. in magnetic resonance imaging.

The present invention relates to macromolecular paramagnetic compounds,to contrast agents containing such compounds and their use in magneticresonance imaging (MRI) of human and non-human subjects, to chelatingagents for use in the manufacture of such compounds and to the use ofsuch chelating agents and chelates and and salts thereof in therapy anddiagnosis.

In MRI, the contrast in the images generated may be enhanced byintroducing into the zone being imaged an agent which affects the spinreequilibration characteristics of the nuclei (the "imaging nuclei",which are generally protons and more especially water protons) which areresponsible for the resonance signals from which the images aregenerated. In this respect it has been found that contrast enhancementresults from the use of contrast agents containing paramagnetic,superparamagnetic or ferromagnetic species. For paramagnetic contrastagents, the enhanced image contrast derives predominantly from thereduction in the spin reequilibration coefficient known as T₁ or as thespin-lattice relaxation time, a reduction which arises from the effecton the imaging nuclei of the fields generated by the paramagneticcentres.

The use of paramagnetic compounds as contrast agents in MRI has beenwidely advocated and a broad range of paramagnetic compounds has beensuggested in this regard. Thus for example Lauterbur and others havesuggested the use of manganese salts and other paramagnetic inorganicsalts and complexes (see Lauterbur et al. in "Frontiers of BiologicalEnergetics", volume 1, pages 752-759, Academic Press (1978), Lauterburin Phil. Trans. R. Soc. Lond. B 289: 483-487 (1980) and Doyle et al. inJ. Comput. Assist. Tomogr. 5(2): 295-296 (1981)), Runge et al. havesuggested the use of particulate gadolinium oxalate (see U.S. Pat. No.4,615,879 and Radiology 147(3): 789-791 (1983)), Schering AG havesuggested the use of paramagnetic metal chelates, for example ofaminopolycarboxylic acids such as nitrilotriacetic acid (NTA),N,N,N',N'-ethylenediaminetetraacetic acid (EDTA),N-hydroxyethyl-N,N',N'-ethylenediaminetriacetic acid (HEDTA),N,N,N',N",N"-diethylenetriaminepentaacetic acid (DTPA) and1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) (see for exampleEP-A-71564, EP-A-130934 and DE-A-3401052), and Nycomed AS have suggestedthe use of paramagnetic metal chelates of iminodiacetic acids (seeEP-A-165728). Many other paramagnetic contrast agents have beensuggested in the literature, for example in EP-A-230893, EP-A-232751,EP-A-292689, EP-A-255471, EP-A-292689, EP-A-287465, U.S. Pat. No.4,687,659 and WO86/02005. Besides the chelates of DOTA and DTPA, thechelates of N,N" (bis methyl-carbamoylmethyl) N,N',N"-diethylenetriaminetriacetic acid (DTPA-BMA),1-oxa-4,710-triazacyclododecane-N,N',N"-triacetic acid (OTTA) andN-[2,3-dihydroxy-N-methyl-propylcarbamoylmethyl]-1,4,7,10-tetraazacyclododecane-N',N",N'"-triaceticacid, etc. (DO3A) deserve particular mention.

Paramagnetic compounds in which the paramagnetic centre is bound in achelate complex have been considered particularly desirable as otherwisetoxic heavy metals, such as gadolinium for example, may in this way bepresented in a biotolerable form. The use of chelating agents, such asEDTA, DTPA, etc., known for their efficacy as heavy metal detoxificationagents has thus received particular attention (see for example Weinmannet al., In AJR 142: 619-624 (1984)).

While the toxicities of the paramagnetic chelates are generally lowerthan those of the inorganic salts of the same paramagnetic metalspecies, the efficiency of such chelate complexes in contrastenhancement is not greatly improved relative to that of the salts.

It has however been found that by binding the paramagnetic species to arelatively heavy carrier, for example a macromolecule, increasedcontrast effect can be achieved, perhaps at least in part due to theeffect of the heavy carrier in slowing down tumbling motions of theparamagnetic species. This is well illustrated by Technicare Corporationin EP-A-136812. Binding macromolecules to paramagnetic compounds hasalso been suggested as a means by which tissue-specific paramagneticcontrast agents can be produced. Thus, for example, Schering AG inEP-A-71564, suggest binding paramagnetic chelates to biomolecules suchas hormones, proteins and the like to cause the contrast agent afteradministration to congregate at particular body sites. TechnicareCorporation, in EP-A-136812, similarly suggest binding paramagnetic ionsto tissue-specific macromolecules such as, for example, antibodies.

Binding paramagnetic chelates to albumin to produce a blood poolingcontrast agent has also been suggested and one such compound, GdDTPA-albumin, is discussed by Schmiedl et al. in Radiology 162:205(1987). Proteins such as albumin are substances of very complicatedstructure and generally possess limited stability. In particular,protein bound substances are difficult to formulate into solutions andshould not be subjected to heat treatment, and thus contrast agentscontaining such substances cannot be sterilized by the application ofheat. Furthermore, to reduce the risk of allergic response it wouldgenerally be appropriate to utilize a human-derived protein, e.g. humanalbumin, and thus a possible risk of viral contamination from the humansource arises. Consequently, Nycomed As, in EP-A-184899 and EP-A-186947,suggested MRI contrast agents comprising paramagnetic chelatesassociated with thermostable, readily characterized, biologicallyrelatively passive macromolecules such as polysaccharides, e.g.dextrans. Thus EP-A-186947 discloses soluble macromolecular paramagneticcompounds which where they have molecular weights above the kidneythreshold may function as blood pooling MRI contrast agents.

Amersham International PLC have also suggested, in WO85/05554, the useof macromolecular carriers for paramagnetic chelates for use as MRIcontrast agents. However, stressing the importance that the chelatecomplex must be stable in vivo (in particular where the paramagneticmetal ion itself is toxic) Amersham have suggested that the possibilityof the macromolecule sterically hindering chelation of the paramagneticmetal species by the chelating entity may be avoided by binding thechelating entity to the macromolecule through the agency of a linkermolecule, for example to produce the compound X--OCONH--(CH₂)_(n)--NHCO--Y, where X is the macromolecule and Y is the chelating entity.One such chelate-linker-macromolecule compound, GdDOTA-glycine-dextran,is also disclosed in EP-A-186947.

Other paramagnetic MRI contrast agents are disclosed in the literature(see for example WO87/02893, U.S. Pat. No. 4,639,365 and WO87/01594 andthe references listed in these documents) and there have been severalreviews of paramagnetic MRI contrast agents (see for example AJR 141:1209-1215 (1983), Sem. Nucl. Med. 13: 364 (1983), Radiology 147: 781(1983) and J. Nucl. Med. 25: 506 (1984)).

When a paramagnetic compound is administered into the cardiovascularsystem of a subject to be imaged, the fate of the compound depends on anumber of factors. If it comprises insoluble particulate matter, it willbe removed from the blood system by the reticuloendothelial system(RES), in particular by Kupffer cells of the liver; if it containsrelatively large particles, such as liposomes, these may lodge in thelungs; and if the compound is soluble and of relatively low molecularweight it may be cleared out of the blood through the kidneys relativelyrapidly (as is the case with GdDTPA-dimeglumine, an agent developed andtested by Schering AG). Thus GdDTPA-dimeglumine has a half life in theblood of about 20 minutes (see Weinmann et al. in AJR 142: 619-624(1984)).

However for a paramagnetic MRI contrast agent to be suitable as a bloodpooling agent, i.e. one which is not rapidly removed from thecardiovascular system, it is necessary that the paramagnetic compound besoluble, that it should have a molecular weight sufficiently high as toprevent rapid excretion through the kidneys, and that it should have anin vivo stability which achieves a balance between the stabilityrequired to ensure adequate half life in the blood pool and theinstability required for the compound, or more particularly theparamagnetic species contained therein, to be excretable.

We have now found that by the use of a linker moiety which is bound tothe macromolecule by an ester grouping and to the chelating moiety by anamide grouping and which provides a carbon chain of at least 2 atoms inlength between the ester and amide groups, it is possible to providemacromolecular paramagnetic MRI contrast agents with improvedproperties, in particular for the imaging of the cardiovascular system.More particularly, we have found that by the use of such linker moietiesa particularly desirable balance between in vivo stability and in vivoinstability is achieved.

Thus in one aspect the present invention provides a paramagneticcompound comprising a paramagnetic metal species chelated by a chelatingmoiety bound by an amide group to a linker group itself bound by anester group to a macromolecule, wherein said linker group provides acarbon chain of from 2 to 11 atoms between said amide group and saidester group.

The linker group in the paramagnetic compounds of the present inventionis preferably the residue of an amino acid of formula I

    HOOC--CH.sub.2 --(CHR).sub.n --NH.sub.2                    (I)

(wherein, n is an integer of from 1 to 10, and each R, which may be thesame or different, represents a hydrogen atom or a hydroxyl,hydroxyalkyl, or C₁₋₄ alkyl group, with the proviso that R on the carbonattached to the amine group does not represent a hydroxyl group).

In formula I above, n is preferably an integer of from 1 to 6, anespecially preferably from 1 to 3, and R is preferably hydrogen, methyl,ethyl, hydroxyl, mono- or poly-hydroxy (C₁₋₆ alkyl), especially mono- orpoly-hydroxy (C₁₋₄ alkyl), for example hydroxymethyl or2,3-dihydroxy-propyl. Where R is a polyhydroxyalkyl group, the ratio ofhydroxyl groups to carbon atoms is preferably up to 1:1. Residues ofcompounds of formula I in which n is from 1 to 10 and R is hydrogen alsoare preferred as the linker group in the paramagnetic compounds of theinvention. Particularly preferred identities for the linker groupinclude the residues of beta and gamma amino acids, for examplebeta-alanine and 4-amino-butanoic acid.

The chelating moiety in the paramagnetic compounds of the presentinvention may conveniently be the residue of a conventional metalchelating agent. Suitable such agents are well known from the literaturerelating to MRI contrast agents discussed above (see for exampleEP-A-71564, EP-A-130934, EP-A-186947, U.S. Pat. No. 4,639,365,EP-A-230893, EP-A-232751, EP-A-292689, EP-A-255471, U.S. Pat. No.4,687,659, WO-86/02005 and DE-A-3401052) as well as from the literaturerelating to chelating agents for heavy metal detoxification.

The chelating moiety chosen should clearly be one that is stable in vivoand is capable of forming a chelate complex with the selectedparamagnetic species. Preferably however, the chelating moiety will beone as described in EP-A-186947 or the residue of an aminopoly(carboxylic acid or carboxylic acid derivative) (hereinafter an APCA) ora salt thereof, for example one of those discussed by Schering AG inEP-A-71564, EP-A-130934 and DE-A-3401052 and by Nycomed AS inInternational Patent Application No. PCT/GB88/00572. This latterapplication discloses APCAs which carry hydrophilic groups, e.g. on theamine nitrogens or on the alkylene chains linking the amine nitrogens,for example compounds of formula II ##STR1## (wherein each of the groupsZ is a group --CHR₁ X or the groups Z are together a group --(CHR₁)₂--A'--(CHR₁)₂, where A' is O, S, N--CHR₁ X or N--(CHR₁)_(p) --N(CHR₁ X)₂where p is 2, 3 or 4; Y is a group --(CHR₁)₂ --N(CHR₁ X)₂ or a group--CHR₁ X; each X, which may be the same or different, is a carboxylgroup or a derivative thereof such as an amide, ester or carboxylatesalt derivative, or a group R₁ ; each R₁, which may be the same ordifferent, is a hydrogen atom, a hydroxyalkyl group or an optionallyhydroxylated alkoxy group; with the proviso that at least two nitrogenscarry a --CHR₁ X moiety wherein X is a carboxyl group or a derivativethereof, and preferably the provisos that each --CHR₁ X moiety is otherthan a methyl group, and that where Y and Z are --CHR₁ X groups at leastone R₁ is other than hydrogen, and preferably also that each nitrogenatom carrying a --CHR₁ X moiety wherein X is a carboxyl group or aderivative thereof carries at least one such moiety which is other thana --CH₂ X moiety) and salts thereof.

Particularly preferred as chelating moieties for the paramagneticcompounds of the present invention are the residues of the following:EDTA; DTPA; OTTA; DO3A; DTPA-BMA; DOTA; desferrioxamine; and thephysiologically acceptable salts thereof, especially DTPA, DOTA andsalts thereof.

Where the chelating moiety in the paramagnetic compounds of the presentinvention has a labile counterion, that counterion should be aphysiologically tolerable ion, for example the ion of an alkali metal, anon-toxic amine (for example tris(hydroxymethyl)aminomethane,ethanolamine, diethanolamine and N-methylglucamine), a halogen, or anon-toxic organic or inorganic acid.

As the macromolecule component of the paramagnetic compound of thepresent invention there can be used any of the macromolecules previouslysuggested for macromolecular paramagnetic MRI contrast agents.Preferably, the macromolecule chosen will be one which isphysiologically tolerable and which contains hydroxyl groups or whichcan be chemically modified to introduce hydroxyl groups or to deprotectprotected hydroxyl groups.

Particularly preferably, the macromolecule will be a hydroxyl groupcontaining material selected from the group consisting of polymeric andpolymerized carbohydrates and polymerized sugar alcohols and derivativesthereof. The term "polymeric carbohydrate" is used to designate anaturally occurring polymer built up of carbohydrate monomers and theterm "polymerized carbohydrate" is used to designate a synthetic polymerobtained by polymerizing carbohydrate molecules, for example with theaid of coupling or cross-linking agents. Similarly the term "polymerizedsugar alcohol" is used to designate a synthetic polymer obtained bypolymerizing sugar alcohol molecules, for example with the aid ofcoupling or cross-linking agents.

The macromolecule may thus conveniently be a cyclic or acyclicpolysaccharide, such as a glucan, for example starch, amylose,amylopectin (including macromolecular dextrins thereof), glycogen,dextran and pullalan, or a fructan, for example inulin and levan,cyclodextrine or other physiologically tolerable polysaccharides ofvegetable, microbial or animal origin.

Examples of polymerized carbohydrates or sugar alcohols which can beused as the macromolecule include so-called polyglucose, which isobtained by polymerization of glucose, and macromolecular productsobtained by cross-linking carbohydrates or sugar alcohols (for examplemannitol or sorbitol) with at least one bifunctional cross-linkingagent, for example epichlorohydrin, a diepoxide or a correspondinghalogen hydrin or with a bifunctional acylating agent. An example ofsuch a product which is commercially available is Ficoll (Ficoll is aTrade Mark of Pharmacia Fine Chemicals AB of Uppsala, Sweden) which isobtained by cross-linking sucrose with the aid of epichlorohydrin.

Further examples of substances which can form the basis for themacromolecule include physiologically tolerable derivatives of thepolysaccharides mentioned above, for example hydroxyl, carboxyalkyl,acyl or alkyl derivatives, for example hydroxyethyl, dihydroxypropyl,carboxymethyl, acetyl and methyl derivatives of such polysaccharides.

Water-soluble derivatives of insoluble polysaccharides (for example ofcellullose) may be considered as well as the water-solublemacromolecules mentioned above. Many such macromolecules arecommercially available and/or are extensively described in theliterature.

Although the paramagnetic compounds of the invention are particularlysuited for use as blood pooling agents when the compounds are solubleand have molecular weights above the kidney threshold, lower molecularweight paramagnetic compounds of the invention may be used in other MRIcontrast agents, e.g. agents for investigation of the kidneys, bladderor gastrointestinal tract.

The macromolecule will generally be chosen according to the intended useof the macromolecular paramagnetic chelate. If for example the chelateis to be used in investigation of body cavities having outward escapeducts, for example the gastrointestinal tract, the bladder and theuterus, the macromolecule need not be biodegradable. Furthermore wherethe chelate is intended for parenteral administration, the macromoleculeagain need not be biodegradable as long as its molecular weight issufficiently small as to allow its excretion into the urine. However,where the chelate is to be used in a blood pooling agent it is desirableeither to use biodegradable macromolecules whose molecular weightsexceed the kidney threshold or to use macromolecular compounds for whicheach molecule contains more than one macromolecule, for examplecompounds having a macromolecule-linker-chelate-linker-macromoleculestructure. Where a biodegradable macromolecule is to be used, these mayfor example be macromolecules which are enzymatically degradable byhydrolyses, for example endohydrolases which hydrolyze glycosidiclinkages in the macromolecule. Thus for example macromoleculesdegradable by alpha-amylase, for example starch-based macromolecules,may be chosen.

The macromolecules used for the paramagnetic compounds of the inventionmay be neutral or may have a net negative or positive charge insolution. For parenteral use, macromolecules with no net charge or witha negative net charge in solution are preferred. A negative net chargemay be obtained for example by introducing carboxyl groups or othernegatively charged groups into the macromolecules if such groups are notalready present therein.

It is particularly preferred that the macromolecule in the compounds ofthe invention be a polysaccharide and especially preferably a dextran ora derivative thereof, particularly are having a weight average molecularweight of from 40,000 to 500,000 especially about 70,000.

The molecular weight of the paramagnetic compounds of the presentinvention can easily be selected to suit the particular end use for thecompound. As indicated above, this may be done either by selection ofappropriately sized macromolecules or by linking together two or moremacromolecules to produce the final compound. For general diagnosticpurposes, the weight average molecular weight of the paramagneticcompound is preferably in the range of 1,000 to about 2,000,000,preferably 3,000 to about 2,000,000. For the preparation of suchparamagnetic compounds , macromolecules of the desired molecular weightcan be obtained by conventional methods.

Where it is desired that the paramagnetic compound should be excretableinto the urine without prior degradation, the molecular weight ispreferably less than 40,000, for example less than 30,000 or moreparticularly less than 20,000. Where however, the paramagnetic compoundsof the present invention are to be used as blood pooling agents, the useto which they are particularly well adapted, the molecular weight of theparamagnetic compound should preferably lie in the range 40,000 to2,000,000, more preferably 60,000 to 100,000. Where the paramagneticcompound comprises a single macromolecule residue, the molecular weightrange limits listed above may be considered to be the appropriate rangelimits for the molecular weight of the macromolecule also.

In the paramagnetic compounds of the present invention, the paramagneticmetal species, i.e. a paramagnetic metal atom or ion, is preferablynon-radioactive and is particularly preferably selected from the groupof elements having atomic numbers 21-29, 42, 44 and 57-71, the elementshaving atomic numbers 24-29 or 62-69 being specially preferred. Examplesof suitable lanthanides include gadolinium, europium, dysprosium,holmium, and erbium and examples of other suitable elements includemanganese, iron, nickel, chromium and copper. The particularly preferredparamagnetic metal species include Cr(III), Mn(II), Fe(III), Dy(III) andGd(III), especially Gd and Dy and Cr.

In a further aspect, the present invention provides a process for thepreparation of the macromolecular paramagnetic compounds of the presentinvention, which process comprises admixing in a solvent an at leastsparingly soluble paramagnetic metal compound, for example a chloride,oxide or carbonate, together with a macromolecular chelating agentcomprising a chelating moiety bound by an amide group to a linker groupitself bound by an ester group to a macromolecule, wherein said linkergroup provides a carbon chain of at least two atoms. between said amidegroup and said ester group.

The macromolecular chelating agent mentioned in the previous paragraphitself represents a further aspect of the present invention.

Thus in a still further aspect the present invention provides amacromolecular chelating compound comprising a chelating moiety bound byan amide group to a linker group itself bound by an ester group to amacromolecule, wherein said linker group provides a carbon chain of atleast two atoms between said amide group and said ester group, or a saltor metal chelate thereof.

The macromolecular chelating agent can itself be prepared by condensinga hydroxyl group containing macromolecule with an amino acid or a saltthereof and reacting the product so obtained with a carboxyl group-, orreactive carboxyl derivative-, containing chelating agent. Thus in a yetstill further aspect the present invention provides a process for thepreparation of a macromolecular chelating agent according to the presentinvention which process comprises: reacting a hydroxyl group containingmacromolecule with an amino acid or a salt thereof, said amino acidhaving a carbon chain of at least two atoms between its carboxyl andamine groups, and conveniently being an amino acid of formula I asdefined above; reacting the product so obtained with a carboxyl group-,or reactive carboxyl derivative-, containing chelating agent; and,optionally, converting the product so obtained into a salt or metalchelate thereof.

Where the paramagnetic compounds of the present invention are to beadministered to the human or non-human animal body as MRI contrastagents, they will conveniently be formulated together with one or morepharmaceutical carriers or excipients. Thus in a further aspect of thepresent invention provides a diagnostic contrast medium comprising amacromolecular paramagnetic compound according to the present inventiontogether with at least one pharmaceutical carrier or excipient.

The chelating agents and the salts and chelates according to theinvention are also useful in other fields in which chelating agents andchelates have been used, for example as stabilizers for pharmaceuticalpreparation, as antidotes for poisonous heavy metal species and asdiagnostic agents for the administration of metal species (e.g. atoms orions) for radiotherapy or for diagnostic techniques such as X-ray, andultrasound imaging or scintigraphy. In addition the paramagneticcompounds may also be useful in techniques such as lymph angiography. Ina further aspect therefore the present invention provides a diagnosticor therapeutic composition comprising at least one pharmaceuticalcarrier or excipient together with a metal chelate whereof the chelatingmoiety is the residue of a chelating compound according to theinvention.

In a still further aspect the present invention also provides adetoxification agent comprising a chelating compound according to theinvention, optionally in the form of a salt or chelate with aphysiologically acceptable counterion, together with at least onepharmaceutical carrier or excipient.

The compositions, e.g. contrast media of the present invention mayinclude conventional formulation aids, for example stabilisers,antioxidants, osmolality adjusting agents, buffers, pH adjusting agents,etc. and may be in forms suitable for parenteral or enteraladministration, for example injection or infusion or administrationdirectly into a body cavity having an external escape duct, for examplethe gastrointestinal tract, the bladder or the uterus. Thus thecompositions of the present invention may be in a conventionalpharmaceutically administration form such as a tablet, capsule, powder,solution, suspension, dispersion, syrup, suppository, etc; however,solutions, suspensions and dispersions in physiologically acceptablecarrier media, for example water for injections, will generally bepreferred.

Where the compositions of the invention contain a chelate of a toxicmetal species e.g. a heavy metal or radioactive metal ion, it may bedesirable to include within the composition a slight excess, e.g. 0.5 to20 mol %, preferably 1 to 10 mol %, of the chelating compound or of aweaker chelate thereof with a physiologically tolerable counterion, e.g.as discussed by Schering AG in DE-A-3640708 (and AU-A-81889/87).

Where the composition is formulated for parenteral administration, forexample where a contrast medium is to be used as a blood pooling agent,a solution in a sterile physiologically acceptable medium, for examplean isotonic or somewhat hypertonic aqueous solution would be preferred.

For MRI examination, the contrast medium of the present invention, if insolution, suspension or dispersion form, will generally contain theparamagnetic metal species at a concentration in the range 1 micromoleto 1.5 mole per liter, preferably 0.1 to 700 mM. The contrast medium mayhowever be supplied in a more concentrated form for dilution prior toadministration. The contrast medium of the invention may conveniently beadministered in amounts of from 10⁻⁴ to 3 mmol e.g. 10⁻³ to 1 mmol ofthe paramagnetic metal species per kilogram of body weight, e.g. about 1mmol Dy/Kg bodyweight.

For X-ray examination the dose of the contrast agent should generally behigher and for scintigraphic examination the dose should generally belower than for MR examination. For radiotherapy and detoxificationconventional doses may be used.

In a yet further aspect, the present invention also provides a method ofdiagnosis practiced on the human or non-human animal body, which methodcomprises administering to said body a macromolecular metal chelate,preferably a paramagnetic compound, according to the present inventionand generating an X-ray, magnetic resonance, ultrasound or scintigraphicimage of at least part of said body.

In a still further aspect the invention provides a method of heavy metaldetoxification practiced on the human or non-human animal body, whichmethod comprises administering to said body a chelating compoundaccording to the invention, optionally in the form of a salt or chelatewith a physiologically acceptable counterion.

In a yet still further aspect the invention also provides a method ofradiotherapy practiced on the human or non-human animal body, whichmethod comprises administering to said body a chelate of a radioactivemetal species with a chelating compound according to the invention.

In a still further aspect, the present invention also provides the useof a macromolecular compound or salt or chelate thereof according to theinvention for the manufacture of a diagnostic agent for use in methodsof image generation, detoxification or therapy practiced on the human ornon-human animal body.

As mentioned above, as a result of the use of the particular linkergroups, the paramagnetic compounds of the present invention haveproperties which are particularly improved relative to those of theprior art compounds.

Thus where the paramagnetic chelate GdDTPA is bound directly to dextran,the resulting compound is not stable either in vivo or in vitro. Onadministration of such a compound, GdDTPA-dextran (molecular weight70,000) to rabbits, no blood pooling effect was observed and the rapidelimination of the gadolinium into the urine that was observed was verysimilar to that which is observed for GdDTPA or its salts. In contrast,GdDTPA linked by beta-alanine to dextran of molecular weight 70,000, acompound according to the present invention, is stable in vitro and hasalmost ideal blood pooling properties insofar as it exhibits a half lifein the blood of about 6 hours and has a distribution volume of 0.05l/kg, a distribution volume which indicates that at least untildegradation the distribution of the compound is essentially only withinthe blood pool.

Nevertheless, the increased blood pooling effect achieved using theamino acid residue linker is not obtained at the expense of readyexcretability of the paramagnetic species due to the presence in theparamagnetic compound between the macromolecule and the linker of anester bond which, unlike the essentially non-biodegradable amide bondsin the macromolecule-linker-chelate compounds of WO-85/05554, isbiodegradable.

The disclosures of all of the documents mentioned herein areincorporated by reference.

The following Examples are provide to illustrate the present inventionin a non-limiting manner. The products of Examples 1 and 14 are howeverparticularly preferred. The following abbreviations are used herein:

Dextran X: Dextran with molecular weight X. 10³ daltons (such dextransare available from Sigma Chemicals)

DMSO-A: dimethylsulfoxide

DTPA-A: diethylenetriamine pentaacetic acid bisanhydride

ECDI: N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide

FMOC-BA: fluorenylmethyloxycarbonyl-beta-alanine PP:4-pyrrolidinopyridine

Water: water deionized by reverse osmosis

EXAMPLE 1 GdDTPA-beta-alanine-dextran (Molecular Weight 70,000)

To a solution of 15.9 g of Dextran 70 in 650 ml of dry DMSO was added20.3 g of FMOC-BA, 13.7 g of ECDI and 968 mg of PP dissolved in 350 mlof dry DMSO. The reaction mixture was stirred at ambient temperature for18 hours and 43.1 g of piperidine was added. After 70 minutes, 7.3 ml ofconcentrated hydrochloric acid was added dropwise, and cooling on anice/water bath and dropwise addition of 1.7 l of an ether/chloroformmixture (7:3 w/w) yielded a yellow oil. After decantation, the oil wasdissolved in distilled water and the pH was adjusted to 4. Sodiumchloride was added until the salt concentration was 0.9% in 1400 ml ofsolution, and the product was dialyzed against 0.9% sodium chloride inwater at pH 4 in a hollow fibre cartridge (Amicon HP 10-20) for 24hours. The solution was then concentrated using the same equipmentagainst distilled water to a volume of 1150 ml, the pH was adjusted to 9with N-methylmorpholine and 29.18 g of DTPA-A was added while the pH waskept at 8 using the same base. When the solution became clear, thereaction mixture was stirred for 2 hours, 43.78 g of citric aciddissolved in 47.4 ml of 10N NaOH was added, and the pH was adjusted to6.0 with concentrated hydrochloric acid. 30.37 g of gadolinium chloridehexahydrate dissolved in 200 ml of distilled water was added quickly andthe pH was adjusted to 5.5 using 10N NaOH. The solution was dialyzedagainst distilled water until the relaxation time T₁ (determined using aNMR Proton Spin Analyzer, RADX Corporation, Houston, Tex., USA, at 10MHz and 37° C.) was above 2000 ms. Lyophilization of the solutionyielded 15.3 g of a light yellow coloured powder.

ANALYSIS

Elemental analysis: Gd 4.6%; N 2.15%; Na 0.16%; Cl less than 0.01%. FreeGd (xylene orange titration), DTPA, GdDTPA, citric acid, or DMSO (HPLC):less than 0.01% (The percentages in the analysis results are by weight).

The specific relaxation rate (T₁) enhancement (SRRE) (measured in an NMRProton Spin Analyzer RADX Corp. Houston, Tex., USA at 10 MHz and 37° C.)in distilled water was 9.6 s⁻¹ mM⁻¹ Gd.

EXAMPLE 2 Injection Solution

78.6 mg of gadolinium (III) DTPA-beta-alanine-dextran (molecular weight70,000) were prepared in accordance with Example 1 and dissolved in 10ml of distilled water. The solution was sterile filtered and filled intoa 10 ml vial. The solution contained 0.05 mmol Gd/ml.

EXAMPLE 3 Pharmacokinetics in Rabbits

The solution of Example 2 was injected intravenously into three rabbitsat a dose of 0.05 mmol Gd/kg body weight. Three other rabbits receivedgadolinium

(III) DTPA-dimeglumine salt intravenously at a dose of 0.05 mmol Gd/kgbody weight.

Blood samples were drawn from an ear vein before injection and at 1, 5,10, 15, 30, 120, 180 and 300 minutes and 24 and 48 hours afterinjection. Serum was prepared from the blood samples and the relaxationtimes T₁ and T₂ were determined in an RADX NMR spectrometer (37° C., 10MHz). The gadolinium concentration in the serum samples were determinedby ICP (inductive coupled plasma). The apparent volume of distribution(V_(D)) and the biological half-life (t_(1/2)) were determined using thetwo-compartment model. The results are as set forth in the Table below:

                  TABLE                                                           ______________________________________                                                          V.sub.D    t.sub.1/2                                        SAMPLE            (l/kg)     (hours)                                          ______________________________________                                        Gd(III)DTPA-beta- 0.05 ± 0.003                                                                           6.7 ± 0.18                                   alanine-dextran 70                                                            Gd(III)DTPA-dimeglumine                                                                         0.26 ± 0.037                                                                          0.72 ± 0.11                                   ______________________________________                                    

The results presented above show the compound of Example 1 to have aconsiderably longer half-life than GdDTPA and yet still to bebiodegradable as no relaxation effects and no serum gadolinium wereobserved in serum 48 hours after injection. The observed apparent volumeof distribution of 0.05 confirms its blood pooling property.

EXAMPLE 4 Dextran 70-beta-alanine-DTPA

10.0 g of Dextran 70 was reacted with 12.8 g of FMOC-BA, 8.7 g of ECDI,0.61 g of PP and 31.5 ml of piperidine in 600 ml of dry DMSO and thenwith 22 g of DTPA-A as described in Example 1 to the point where thecitric acid buffer was added. The pH was adjusted to 5.1 and 6M HCl andthe solution was dialyzed against 4 l of water. The solution waslyophilized to give 5.5 g of a light yellow solid.

Elemental analysis: N 2.82%, C 42.52%, H 6.74%.

EXAMPLE 5 Dextran 70-beta-alanine-DTPA-Fe (III)

0.5 g of the product of Example 4 was dissolved in 70 ml of water and tothis were added 1.38 g of citric acid, 1.49 ml of 10N NaOH and 417 mg ofFeCl₃ dissolved in 10 ml of water. The pH was adjusted to 5 with 10NNaOH and, after reaction overnight, the solution was dialyzed againstwater until T₁ in the filtrate was above 2000 ms. Lyophilization gave0.47 g of a light brown solid, 5.5% Fe, relaxivity 0.8 s⁻¹ mM⁻¹.

EXAMPLE 6 Dextran 70-beta-alanine-DTPA-Dy

0.5 g of the product of Example 4 was complexed with 1.1 g of DyCl₃ andisolated as described in Example 5. Yield 0.57 g of a white solid, 9.8%Dy, relaxivity 0.2 s⁻¹ mM⁻¹.

EXAMPLE 7 Dextran 70-beta-alanine-DTPA-Yb

0.5 g of the product of Example 4 was complexed with 1.15 g of Yb(NO₃)₃and isolated as described in Example 5. Yield 0.5 g of a yellowishsolid, 3.5% Yb, relaxivity 0.03 s⁻¹ mM⁻¹.

EXAMPLE 8 Dextran 70-beta-alanine-DTPA-Cu

0.5 g of the product of Example 4 was complexed with 642 mg of CuSO₄ andisolated as described in Example 5. Yield 0.55 g of a light blue solid,1.5% Cu, relaxivity 0.3 s⁻¹ mM⁻¹.

EXAMPLE 9 Dextran 40-beta-alanine-DTPA-Gd

2.0 g of Dextran 40 was reacted with 2.6 g of FMOC-BA, 1.73 g of ECDI,122 mg of PP and 6.3 ml of piperidine in dry DMSO as described inExample 1. The product was reacted further with 3.77 g of DTPA-A asdescribed herein, and after complexation in citrate buffer with 3.82 gof GdCl₃. 6 H₂ O the product was dialysed and lyophilized to yield 1.05g of a white solid, 5.1% Gd, relaxivity 5.1 s¹ mM⁻¹.

EXAMPLE 10 Hydroxyethylstarch-beta-alanine-DTPA-Gd

2.0 g of hydroxyethylstarch (prepared by hydroxyethylation of waxystarch with ethylene oxide according to the method described in U.S.Pat. No. 2,516,634) molecular weight 131,000 and degree of substitution0.52, was dissolved in 120 ml of dry DMSO. It was reacted with the samereagents in the same quantities and isolated as described in Example 9.Yield 2.3 g of white solid, 5.1% Gd, relaxivity 6.0 s¹ mM⁻¹.

EXAMPLE 11 Dextran 40-beta-alanine-EDTA-Cr

2.0 g of Dextran 40 was reacted as described in Example 9 up to thepoint where the Dextran 40-beta-alanine water solution had been dialysedat pH 4.2.2.65 g of EDTA-bis-anhydride (prepared using the method ofEckelman et al, J Pharm. Sci., 64 (1975) 704) was reacted with theDextran-derivative, complexed with 2.74 g of CrCl₃. 6 H₂ O and theproduct was isolated as described in Example 9. Yield 2.9 g of a purplesolid, 3.1% Cr, relaxivity 1.1 s⁻¹ mM⁻¹.

EXAMPLE 12 Dextran 500-beta-alanine-DTPA-Bi

2.0 g of Dextran 500 was reacted as described in Example 9 except that4.4 g of DTPA-A was used. The reaction mixture was stirred for 3 hourswhile the pH was kept at 8 with N-methylmorpholine. The pH was thenadjusted to 5 with 6N HCl and a buffer solution containing 5.5 g ofcitric acid and 5.96 ml of 10N NaOH was added. A solution of Bi(III) wasprepared by dissolving 3.89 g of BiCl₃ in 100 ml of 1M HCl and the pHwas adjusted to 7 with saturated ammonia in water. The suspension wascentrifuged and the supernatant was decanted off. The precipitate wasresuspended and centrifuged twice and the white jelly-like precipitatewas added to the buffer solution of the Dextran. The pH was 5.0 and,after reaction overnight, the clear solution was dialysed against 12 lof water and lyophilization yielded 0.8 g of a white solid, 9.4% Bi.

EXAMPLE 13 Dextran 2000-beta-alanine-HEtDTPA-Gd

(a) The DTPA-derivative3,6,9-tris-carboxymethyl-4-(2-hydroxyethyl)-3,6,9-triazaundecane diacid(HEtDTPA) was synthesized according to the method of PCT/GB88/00572.HEtDTPA trihydrochloride was prepared by loading HEtDTPA on a stronganion ion exhanger and eluting with 1M HCl followed by evaporation. Theproduct was a white solid, mp. greater than 350° C. (decomp.).

Elemental Analysis: Calc.: C 35.14%, H 5.54%, N. 7.69%, Cl 19.45%.Found: C 34.76%, H 5.46%, N 7.74%, Cl 19.56%.

(b) 2.0 g of Dextran 2000 was reacted as in Example 9 to the pointbefore reaction DTPA-A. The solution was lyophilized to yield 1.9 g of awhite solid. The product was dissolved in 200 ml of dry DMSO and therewere added 2.24 g of HEtDTPA trihydrochloride, 0.86 g of ECDI and 35 mgof PP. After stirring for 24 hours the solution was added to a mixtureof 300 ml of ether and 125 ml of CHCl₃. The product was isolated bydecantation of the supernatant. The product was dissolved in 120 ml ofwater and the pH was adjusted to 5 with 10N NaOH. To the solution wasadded a buffer, containing 5.5 g of citric acid and 5.96 ml of 10N NaOH,and then 1.53 g of GdCl₃. 6 H₂ O dissolved in 10 ml of water. The pH wasadjusted to 5 with 10N NaOH and after 3 hours the product was dialyzedand isolated as described in Example 9. Yield 2.5 g of a light brownsolid, 5.7% Gd, relaxivity 1.4 s⁻¹ mM⁻¹.

EXAMPLE 14 Dextran 70-beta-alanine-DOTA-Gd (a)N',N",N"N'"-Tetracarboxymethyl-1,4,7,10-tetraazacyclododecane (DOTA)

5.26 g of 1,4,7,10-tetraazacyclododecane (prepared as described byStetter et al. Tetrahedron, 37(1981)767) was dissolved in 50 ml ofwater. The pH was adjusted to 10 with conc. HBr, 20.16 g of bromoaceticacid was dissolved in 7 ml of water and a solution of LiOH carefullyadded with cooling in an ice/water bath. The bromoacetic acid lithiumsolution was added to the 1,4,7,10-tetraazacyclododecane solution in oneportion. The pH was kept between 8 and 9.5 with 4N LiOH while thetemperature was gradually increased to 80° C. during 4 hours. Aftercooling, the solution was mixed with 494 ml of wet Dowex 50WX 4 acidicion exchange resin in 1.5 l of water and stirred for 1 hour. Afterthorough washing with water, the gel was washed with 2×750 ml ofsaturated ammonia. The filtrate was evaporated to yield 10.9 g of awhite solid, mp greater than 350° C., FAB-ms M+1 411 and 417 -mono- anddi-lithium salt. ¹³ C-and ¹ H-NMR confirmed the structure. 8.72 g of thesolid was dissolved in 16 ml of water and the pH was adjusted to 2.5with conc. HCl. The white solid was filtered off and the processrepeated with the evaporated filtrate. The collected solids were driedto yield 4.5 g of a white solid, mp greater than 350° C. (decomp).

(b) Dextran 70-beta-alanine-DOTA-Gd

2.0 g of Dextran 70 was reacted as described in Example 9 to the pointbefore DTPA-A was reacted. The product was lyophilized and dissolved in100 ml of dry DMSO. 1.66 g of the precipitated DOTA, 0.86 g of ECDI and62 mg of PP were added and the reaction mixture was stirred overnight atambient temperature. To the reaction mixture was added a mixture of 150ml of ether and 62 ml of CHCl₃, the white precipitate was isolated bydecantation and washing with ether and then dissolved in 80 ml of water.The pH was adjusted to 5 with 10N NaOH and there was added a mixture of5.5 g of citric acid and 5.96 ml of 10N NaOH and then 0.766 g GdCl₃.6H₂O. The reaction mixture was stirred for 50 hours and the product wasisolated by dialysis and lyophilization. Yield 2.4 g of a white solid,7.4% Gd, relaxivity 11.7 s⁻¹ mM⁻¹.

EXAMPLE 15 Dextran70-5-aminopentanoic acid-DTPA-Gd

5.65 g of 9-fluorenylmethyloxycarbonyl-5-amino-valeric acid (preparedfrom 5-amino-valeric acid and 9-fluorenylmethyl chloroformate asdescribed by Carpino et al. J. Org. Chem., 37 (1972)3404) was reactedwith 2.0 g of Dextran 70, 3.5 g of ECDI and 0.25 g of PP as described inExample 9. The reaction mixture was treated with 12.75 ml of piperidine,the product was isolated and dissolved in water and reacted with 7.44 gof DTPA-A as described herein. The product was complexed with 7.74 g ofGdCl₃.6H₂ O in citrate buffer and isolated by dialysis andlyophilization as described above. Yield 6.6 g of a light brown solid,11.4% Gd, relaxivity 5.6 s⁻¹ mM⁻¹.

EXAMPLE 16 Glycogen-beta-alanine-DTPA-Gd

2.0 g of bovine liver glycogen (Sigma Chemicals) was reacted andisolated as described in Example 9. Yield 2.7 g of a white solid, 7.5%Gd, relaxivity 6.8 s⁻¹ mM⁻¹.

EXAMPLE 17 Vial containing Dextran70-beta-alanine-DTPA

A vial is filled with 20 mg of Dextran70-beta-alanine-DTPA (Example 4)and 0.2 mg of Sn(II)Cl₂ as a dry solid.

A solution of ^(99m) Tc as pertechnetate is 0.9% sterile sodium chlorideshould be added before use. The technetium chelate withDextran70-beta-alanine-DTPA is for intravenous or subcutaneousadministration and is a contrast agent for the vascular system or forlymphangiography.

EXAMPLE 18 Dextran70-beta-alanine-DTPA-Gd and the Calcium-disodium saltof Dextran70-beta-alanine-DTPA

760 mg of Dextran 70-beta-alanine-DTPA (Example 4) was dissolved in 10ml water and 28 mg of Ca(OH)₂ were added. The pH was adjusted with NaOHunder ambient conditions. 1 ml of the resulting solution was added to asolution of 1.0 g of Dextran 70-beta-alanine-DTPA-Gd (Example 1) in 9 mlof water, and the resultant solution was sterile filtered, filled into a20 ml vial and lyophilized.

EXAMPLE 19 Dextran70-beta-alanine-DTPA-Gd and the calcium-trisodium saltof DTPA

To a solution of 1.0 g of Dextran70-beta-alanine-DTPA-Gd (Example 1) in10 ml of water was added 17 mg of the calcium-trisodium salt of DTPA(Fluka). The solution was sterile filtered, filled into a 20 ml vial andlyophilized.

We claim:
 1. A paramagnetic compound comprising a paramagnetic metalspecies chelated by a chelating moiety bound by an amide group to alinker group itself bound by an ester group to a macromolecule selectedfrom the group consisting of polymeric and polymerised carbohydrates andpolymerised sugar alcohols, and physiologically tolerable derivativesthereof, wherein said linker group provides a carbon chain of from 2 to11 atoms between said amide group and said ester group.
 2. A compound asclaimed in claim 1 wherein said linker group is a residue of an aminoacid of formula I

    HOOC-CH.sub.2 -(CHR).sub.n -NH.sub.2                       (I)

wherein, n is an integer of from 1 to 10, and each R, which may be thesame or different, represents a hydrogen atom or a hydroxyl, C₁₋₆hydroxyalkyl, or C₁₋₄ alkyl group, with the proviso that R on the carbonattached to the amine group does not represent a hydroxyl group.
 3. Acompound as claimed in claim 2 wherein in formula I n is an integer offrom 1 to 10 and R represents a hydrogen atom.
 4. A compound as claimedin claim 1 wherein said linker group is a residue of a beta or gammaamino acid.
 5. A compound as claimed in claim 1 wherein said linkergroup is a residue of beta-alanine.
 6. A compound as claimed in claim 1wherein said chelating moiety is a residue of an amino polycarboxylicacid or polycarboxylic acid derivative.
 7. A compound as claimed inclaim 1 wherein said chelating moiety is a residue of a compound offormula II ##STR2## wherein each of the groups Z is a group --CHR₁ X orthe groups Z are together a group --(CHR₁)₂ --A'--(CHR₁)₂ --, where A'is O, S, N--CHR₁ X or N--(CHR₁)_(p) --N(CHR₁ X)₂ where p is 2,3 or 4;Yis a group --(CHR₁)₂ --N(CHR₁ X)₂ or a group --CHR₁ X; each X, which maybe the same or different, is a carboxyl group or an amide, ester orcarboxylate salt derivative thereof or a group R₁ ; each R₁, which maybe the same or different, is a hydrogen atom, a hydroxyalkyl group or anoptionally hydroxylated alkoxy group; with the proviso that at least twonitrogens carry a --CHR₁ X moiety wherein X is a carboxyl group or aderivative thereof as defined above, or a salt thereof.
 8. A compound asclaimed in claim 1 wherein said chelating moiety is a residue of acompound selected from DTPA, DOTA, and salts thereof.
 9. A compound asclaimed in claim 1 wherein said macromolecule is a polysaccharide.
 10. Acompound as claimed in claim 1 wherein said macromolecule is a dextranor a derivative thereof.
 11. A compound as claimed in claim 10 whereinsaid macromolecule has a weight average molecular weight of from 40,000to 500,000.
 12. A compound as claimed in claim 1 wherein saidparamagnetic metal is of atomic number 21-29, 42, 44 or 57-71.
 13. Acompound as claimed in claim 12 wherein said paramagnetic metal is Gd,Dy or Cr.
 14. A process for the preparation of a macromolecularparamagnetic compound as claimed in claim 1 which process comprisesadmixing in a solvent an at least sparingly soluble paramagnetic metalcompound, together with a macromolecular chelating agent comprising achelating moiety bound by an amide group to a linker group itself boundby an ester group to a macromolecule selected from the group consistingof polymeric and polymerised carbohydrates and polymerised sugaralcohols, and physiologically tolerable derivatives thereof, whereinsaid linker group provides a carbon chain of from 2 to 11 atoms betweensaid amide group and said ester group.
 15. A macromolecular chelatingcompound comprising a chelating moiety bound by an amide group to alinker group itself bound by an ester group to a macromolecule selectedfrom the group consisting of polymeric and polymerised carbohydrates andpolymerised sugar alcohols, and physiologically tolerable derivativesthereof, wherein said linker group provides a carbon chain of from 2 to11 atoms between said amide group and said ester group, or a salt ofmetal chelate thereof.
 16. A process for the preparation of a chelatingcompound as claimed in and claim 15 which process comprises reacting ahydroxyl group containing macromolecule with an amino acid or a saltthereof, said amino acid having a carbon chain of at least two atomsbetween its carboxyl and amine groups; reacting the product so obtainedwith a carboxyl group-, or reactive carboxyl derivative-containingchelating agent; and, optionally, converting the product so obtainedinto a salt or metal chelate thereof.
 17. A compound as claimed in claim1 which is GdDTPA-beta-alanine-dextran.
 18. A method of imagegeneration, which method involves administering to a human or non-humananimal body an effective amount of a macromolecular metal chelate asclaimed in claim 1 and generating a magnetic resonance, image of atleast part of said body.